The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT

Abstract

Virtually all diuse large cell lymphomas and a signi®cant fraction of follicular lymphomas contain translocations and/or point mutations in the 5' noncoding region of the putative oncogene BCL-6, that are presumed to deregulate its expression. BCL-6 encodes a Cys 2 -His 2 zinc ®nger transcriptional repressor with a POZ domain at its amino-terminus. The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions. In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6. Using a yeast two-hybrid screen, we identi®ed N-CoR and SMRT as BCL-6 interacting proteins. Both N-CoR and SMRT, which were originally identi®ed as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases. We show that the interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay. The POZ domain is necessary and sucient for interaction with these corepressors. BCL-6 and N-CoR co-localize to punctate regions of the nucleus. Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT. We ®nd, in vitro, that POZ domains from a variety of other POZ domaincontaining proteins, including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R, also interact with varying anities with N-CoR and SMRT. We ®nd that BCL-6 POZ domain mutations that disrupt the interaction with NCoR and SMRT no longer repress transcription. In addition, these mutations no longer self associate suggesting that self interaction is required for interaction with the co-repressors and for repression. More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins. Our demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression.

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci

“…Interestingly, the Znfg derivative is nuclear (Figure 1c), indicating that the zinc ®nger region contains at least one of the nuclear targeting signals of BCL6. In contrast to full length BCL6fg, Znfg does not produce regular spherical nuclear substructures ( Figure 1c; Dhordain et al, 1995;Huynh and Bardwell, 1998), but a di use nuclear staining sometimes superimposed by irregularly shaped nuclear areas of stronger concentration ( Figure 1c). As expected, Znfg binds very e ciently to the B6BS probe (Chang et al, 1996; data not shown) but fails to repress via this sequence ( Figure 2).…”

“…The induced UTA-L2 and UTA-Z2 cells gave a pattern indistinguishable from that obtained in induced UTA-L1 cells and induced UTA-Z1 cells, respectively (not shown). Right: the same cultures were stained with DAPI to visualize the nuclei Huynh and Bardwell, 1998). Note that in UTA-L1 or UTA-L2 undergoing mitosis, the BCL6fg staining gives a di use cytoplasmic and nuclear signal superimposed by dots in both compartments (Figure 1c).…”

“…Previous studies have reported that both the POZ domain and the central region between the POZ domain and the zinc ®nger display repressing activity and contribute to the recruitment of the HDACcontaining repressing complex (Albagli et al, 1996;Chang et al, 1996;Baron et al, 1997;Dhordain et al, 1997Huynh and Bardwell, 1998;Wong and Privalsky, 1998). Thus, to determine whether the possible e ects of BCL6fg on cell proliferation could be correlated with its transcriptional silencing activity, we also isolated UTA-6 cells stably transfected with a truncated version of BCL6 (BCL6D1-514fg, thereafter referred to as Znfg) which codes for an entirē agged-zinc ®nger region of BCL6 but lacks its repressing domains.…”

“…Five mg of total RNA were harvested after 48 h, loaded and hybridized with a probe Growth suppression and apoptosis by BCL6 (LAZ3) O Albagli et al tion at least in part by locally inducing a repressive (hypoacetylated) chromatin structure. This mechanism appears to be shared by other POZ/zinc ®nger (POK) transcriptional repressors since PLZF, a relative of BCL6 involved in certain cases of acute promyelocytic leukemia, recruits the same SIN3/ SMRT(NcoR)/HDAC complex and also depends upon HDAC activity to repress transcription (Dhordain et al, 1997;Hong et al, 1997;David et al, 1998;Dhordain et al, 1998;Grignani et al, 1998;Guidez et al, 1998;Lin et al, 1998;Wong and Privalsky, 1998;Huynh and Bardwell, 1998). BCL6 is expressed in most tissues and in many cell types in vitro (Kerckaert et al, 1993;Fukuda et al, 1995;Allman et al, 1996;Albagli et al, 1998).…”

“…In addition, BCL6 dampens Th2 cell mediated immunity. Finally, it binds the STAT cognate sequences and represses transcription activated by STAT-6, one of the central mediators of IL4-driven Th2 cell di erentiation (Dent et al, 1997;Ye et al, 1997;Fukuda et al, 1997;Huynh and Bardwell, 1998). Given the mature B cell origin of DLCL, all of these data provide important insights into the understanding of the implication of BCL6 in B-cell malignancies but do not clarify its role at a cellular level.…”